Field of the Invention
The present invention concerns methods and compositions for treating human immunodeficiency virus (HIV), either prophylactically or post-infection. The compositions and methods relate to a T20 minicircle or vaccines comprising a T20 minicircle. A novel T20 minicircle construct is provided, comprising the nucleic acid sequence of SEQ ID NO:1. The T20 minicircle may be used for anti-HIV therapy (e.g., enfuvirtide), gene therapy or viral eradication in infected hosts, or for production of anti-HIV vaccines or vaccine enhancement in non-infected hosts. T20 may be used alone or in combination with one or more anti-HIV agents, as discussed below. In some embodiments T20 may be expressed as an unconjugated peptide. Alternatively a fusion protein or peptide comprising T20 attached to another therapeutic peptide may be used. All such peptide products may be expressed from a minicircle as disclosed herein, or the minicircle itself may be utilized for therapeutic purposes. Enfuvirtide has been used for salvage therapy in patients with multi-drug resistant HIV, alone or in combination with other anti-viral agents. In certain embodiments, adjuvant agents may be utilized to increase the immune response to T20 or other antigens. An exemplary antigen is a DDD (dimerization and docking domain) peptide, as disclosed herein.
Description of Related Art
Minicircles are episomal DNA vectors that are produced as small (˜4 kb) circular expression cassettes, derived from plasmids but devoid of any prokaryotic DNA (see, e.g., Wikipedia “Minicircle”). Minicircles have been used as transgene carriers for genetic modification of mammalian cells (Id.). Their smaller molecular size enables more efficient transfections and offers sustained expression over a period of weeks as compared to standard plasmid vectors that only work for a few days (see, e.g., Kay et al., 2010, Nature Biotech 28:1287-89). Thus, minicircles may be used for direct transfection of host cells, or may be used for production of cloned peptides which in turn may be used as therapeutic agents and/or for vaccination. Since minicircles contain no bacterial DNA sequences, they are less likely to be recognized by the host immune system and destroyed (Argyros et al., 2011, J Mol Med (Berlin) 89:515-29). They are therefore more suitable for in vivo use than standard expression systems, although performance for in vitro use is also improved (Argyros et al., 2011, J Mol Med (Berlin) 89:515-29).
Minicircle production typically involves production of a “parental plasmid” and induction of the activity of a site-specific recombinase to excise the prokaryotic vector sequences (Chen et al., 2003, Mol Ther 8:495-500). The resulting minicircles may be recovered by a variety of techniques. Early versions of minicircles lacked an origin of replication and were therefore lost as cell division occurred. More recently, self-replicating minicircles comprising an S/MAR (scaffold/matrix attachment region) element have been designed (e.g., Argyros et al., 2011, J Mol Med 89:515-29). Self-replicating minicircles may allow prolonged peptide expression from transfected cells. A need exists for improved minicircle vectors for more effective therapeutic use.
T20 (enfuvirtide) is the first HIV fusion inhibitor approved for therapeutic human use (Morris & Kraus, 2005, J Pediatr Pharmacol Ther 10:215-470. HIV-1 infection is initiated by binding of the gp120 envelope protein to its CD4 cell surface receptor and a coreceptor (CXCR4 or CCR5) (Martinez-Munoz et al., 2014, Proc. Natl Acad Sci USA 111:E1960-69). Binding of gp120 is followed by the release of and conformation changes in the associated viral transmembrane gp41 subunit, which is required for membrane fusion with the host cell (Cai et al., 2011, Curr Top Med Chem 11:2959-84). Enfuvirtide works by targeting a conformational transition in gp41, preventing creation of an entry pore for the viral capsid (Cai et al., 2011, Curr Top Med Chem 11:2959-84). Because it is a peptide with poor oral availability, enfuvirtide is typically administered by subcutaneous injection, after reconstitution by the patient. Due to the chronic nature of the treatment, the difficulty of administration contributes to poor patient compliance (Home et al., 2009, AIDS Res Ther 6:2). A need exists for better T20 vectors that will allow improved means of T20 administration.